PROJECT SUMMARY The therapeutic applications of nucleic acids (NAs) have strong potential for a wide range of disease targets but major obstacles to clinical application include short-lived silencing effect and challenges in delivery to specific cells and tissues. Developing a delivery platform for targeted and stabilized intracellular delivery of RNAi mediating agents to specific tissues is essential to capitalizing on the therapeutic potential that NA-based technologies hold for translational human medicine. As many allergens and viral and bacterial pathogens invade the body via mucosal tissues, these sites represent an important target for delivery of NA-based therapeutics. Our transkingdom delivery platform is characterized by nonpathogenic bacteria that constitutively generate shRNAs for targeted intracellular delivery to mucosal epithelial cells and RNAi silencing without genomic integration. This platform theoretically overcomes challenges with delivery to specific cells and tissues. However, very few tissue types have been treated with this delivery vehicle and the short-lived RNAi effect is still problematic. To build upon our existing NA delivery platform technology and the success it has demonstrated to date, we propose here to improve the RNAi properties of the platform for a prolonged silencing effect and demonstrate this delivery platform's ability to target different tissues relevant to a wide range of clinical applications. Specifically, we will individually evaluate the shRNA-stabilizing effects of a constitutively expressed dsRNA binding protein and methyltransferase, and an RNase R mutant. Our specific aims are to 1) Improve the shRNA stability and the RNAi silencing effects of the parent delivery platform through three approaches to be evaluated in vitro, 2) Test the delivery feasibility, safety, biodistribution, immunogenicity, toxicity, and RNAi silencing effect of each variant in mucosal epithelial tissues in vivo, and 3) Evaluate the therapeutic application of the delivery vehicle variants in an established in vivo disease model. To accomplish this research, we have assembled a multidisciplinary team with specialized experience in RNAi- based antiviral development, development of molecular methods, RNA regulation and metabolism, pre-clinical and clinical drug development, and translational research. If the proposed aims are achieved, this work will provide proof of concept for a therapeutically viable NA delivery platform, and will highlight the feasibility of utilizing these transkingdom RNAi delivery vehicles to target clinically relevant mucosal epithelial tissues for a broad range of disease applications. Our unique approach provides a new perspective on NA delivery and addresses many of the shortcomings associated with current NA delivery approaches. Our delivery platform combines ingenuity with practicality and seeks to transform the field of NA therapeutics and RNAi into a clinical reality.